![]() Avoid using EDTA in your tissue dissociation buffers.You can use a hemocytometer, but we'll likely still do our own count and viability assessment using a Luna FL instrument. Do not use a flow cytometer for counting your cells.Regardless of whether you use bead-based purification or FACS, you should carry out a dead cell removal using this kit (or a similar one).It's OK if purity isn't great, since you'll get separation of cell populations in the scRNA-seq Bead-based purification of cells is preferred over FACS, because it is shorter and usually results in happier cells.This is be fine for flow cytometry, but may damage cells and cause problems for scRNA-seq. bashing up lymph nodes with a rubber policeman). You can purchase ACK here, or make your own using the recipe below: Avoid long incubations or RT incubations with this buffer. If there are still visible RBCs present after pelleting the cells, you should repeat the lysis step. Red blood cell (RBC) lysis should be carried out with ACK buffer on ice for ~5-7 minutes, with gentle pipetting up and down and a brief vortex periodically to help this process along.Stay away from BSA if possible. It can be quite dirty (unless you splurge on the super clean stuff ). Instead, you can use double filtered, heat-inactivated FBS for a protein source in your buffers/media.Buffers should be pre-chilled on ice. Centrifuges should be pre-chilled to 4deg. This will also help prevent cell clumping. Keep everything cold, cold, cold. Cells should always be kept on ice.even when walking from the bench to the centrifuge.If you need a starting point for tissue dissociation protocols, use the Worthinton guide.See this paper for the method, and a comparison of these two digestions when cells are analyzed by scRNA-seq. If you are accustomed to digesting tissues at 37✬, consider switching to a 30min digestion at 6✬ with a cold active protease.If you need to stabilize tissue for up to 48hrs before dissociating into a single cell suspension, the MACS Tissue Storage Solution (Cat # 130-100-008) from Miltenyi Biotec is reported to be ideal for scRNA-seq studies.scRNA-seq is far more sensitive to dead, dying and stressed cells than are bulk functional assays or antibody-based staining experiments. Cell isolation protocols optimized for flow cytometry or in vitro functional assays often are insufficient for scRNA-seq experiments.Although you'll be processing your sample(s) before coming to our lab for encapsulation, here are some guidelines that we and our collaborators have found are critical for generating cell suspensions from tissues. The quality and viability of your cells will have a major impact on the success of the scRNA-seq experiment.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |